Method and apparatus for producing frozen particles using an entrapment zone of atomized cryogenic liquid droplets

ABSTRACT

This invention relates to an apparatus and process for producing frozen particles of a liquid product having a liquid product nozzle for introducing liquid product droplets to be frozen and a plurality of cryogenic nozzles for introducing a cryogenic liquid directed away from the liquid product droplets.

This is a continuation of application Ser. No. 08/049,819 filed Apr. 20,1993 now abandoned.

FIELD OF THE INVENTION

This invention relates to an apparatus and method for producing frozenparticles of a liquid and, more particularly, to an apparatus having aliquid nozzle for introducing liquid droplets to be frozen and aplurality of flat spray cryogenic nozzles for creating an entrapmentzone of atomized droplets of cryogenic fluid so as to freeze the liquiddroplets to produce frozen particles.

BACKGROUND ART

The formation of physically uniform and chemically homogeneous sphericalfrozen particles from aqueous biological solutions and suspensions isessential to the manufacture of unit dose tablets for the pharmaceuticaland diagnostic industries. Physically uniform particles minimizevariability in bulk density and provide for tablet manufacturingprocesses that can produce tablets which vary in weight by no more thanabout one percent. Homogeneous chemical composition of the particles isneeded to insure an accurate unit dose. From a manufacturing perspectiveit is desirable for frozen particles which are processed in varioustypes of manufacturing tablet making equipment to exhibit the free flowproperties characteristic of spherical particles. Furthermore, anymanufacturing process for obtaining frozen particles with these desiredcharacteristics should operate efficiently to offset the generally highcost of reagents and other raw materials.

Known techniques used in processing granular products include dryblending, direct compression, wet granulation, particle fabrication bycoating product nuclei with discrete ingredient, fluidized bedgranulation and spray freezing of frozen particles.

Spray freezing methods generally produce highly uniform particles as aresult of their characteristically rapid freezing processes. However,spray freezing is generally an expensive technique due to the high costof refrigerants and the relatively large product losses which can occur.

Typical methods of spray freezing include striking or impinging asolution to be frozen into or against a liquid refrigerant stream(s) orspray(s), spraying a solution to be frozen onto the surface of a bath ofliquid refrigerant, and spraying solution to be frozen through afreezing zone. Examples of refrigerants typically used to accomplishthis freezing include liquid nitrogen, liquid carbon dioxide, andfluorocarbons. Some of the disadvantages of these known methods are:insufficient throughput to support manufacturing sale needs, loss ofproduct due to partial freezing and/or accumulation on the sides of thefreeze chamber, and inefficient uses of cryogenic liquids.

German Patent No. 2,556,790 filed on Nov. 20, 1972 and published Jun. 6,1974 discloses a process for deep freezing aqueous extracts with afreezing vessel having a conical lower section which provides for theimpingement of a liquid product with a liquid refrigerant. Impingementoccurs by one of three disclosed variations including downwardlyspraying product and upwardly spraying refrigerants, upwardly sprayingboth product and liquid refrigerant, and both upwardly and downwardlyspraying the product and upwardly spraying the liquid refrigerant. Thedisadvantage of this apparatus is the likelihood that frozen productwill adhere to the liquid refrigerant nozzles and the funnel shapedbottom of the spray chamber, thus resulting in yield losses.

U.S. Pat. No. 4,952,224, issued Aug. 28, 1990, discloses an apparatushaving a downwardly directed spray of atomized liquid fat and a radiallyinwardly directed plurality of individual spray jets of liquid nitrogenor carbon dioxide which surround and impinge against the downwardlydirected spray of liquid fat droplets. The individual spray jets of thisapparatus are likely to produce non-uniform agglomerations of theproduct due to the high velocity contact of the liquid refrigerant andthe product. In addition, due to the limited time that the product is incontact with the liquid refrigerant, at least some product is unlikelyto be frozen.

Italian Patent No. 2,659,546 issued Jul. 18, 1985, discloses anapparatus for freezing food products having an insulated freezingchamber with a conical lower section and a liquid refrigerant nozzlewith a vertically downward directed spray located in the top center ofthe chamber. A plurality of product nozzles are distributed around anddirected at the sides of the central refrigerant spray. Using thisapparatus it is likely that unacceptably high particle size variationswould occur during a continuous manufacturing process due to variationslikely to occur in product supply and control systems, such as pressurechanges in the product supply pump and pulsations within the nozzles.

There is needed an apparatus and process for producing uniformly,shaped, homogeneous frozen particles of a liquid, capable of supportingfull scale manufacturing needs, which minimizes product yield losses dueto accumulation of product on the inside of the freeze chamber andpartially frozen particles.

SUMMARY OF THE INVENTION

Many of the disadvantages of the prior art are solved by the apparatusand process of the present invention. The apparatus and method of thepresent invention allow for the production of physically uniform andchemically homogeneous particles in a way that maximizes throughput orproduct yield and minimizes loss of product due to partial freezingand/or accumulation on the inner walls of the apparatus, and furtherminimizes the efficient use of cryogenic liquid. By physically uniformis meant of substantially the same size, shape and density.

This invention relates to an apparatus for utilizing a cryogenic liquidfor producing frozen particles of a liquid product which comprises:

(a) a housing;

(b) means including at least three cryogenic liquid nozzles forproducing a substantially continuous, annular downwardly directedcircumferential wall of cryogenic liquid defining an interior entrapmentzone; and

(c) a liquid product nozzle positioned in the housing for introducingdroplets of the liquid product into the entrapment zone whereby thecryogenic liquid freezes the liquid product droplets to produce frozenparticles.

Yet another aspect of the invention relates to a process for utilizing acryogenic liquid for producing frozen particles of a liquid product in ahousing which comprises the steps of:

(a) introducing the cryogenic liquid into the housing in an annular,downward direction creating an substantially continuous downwardlydirected circumferential wall of cryogenic liquid, defining an interiorentrapment zone; and

(b) introducing droplets of the liquid product into the entrapment zone,

whereby the cryogenic liquid freezes the liquid product droplets toproduce frozen particles.

The apparatus and process of the present invention offer significantadvantages over the prior art by providing highly uniform, homogeneousfrozen particles with minimum process yield losses. Yield losses areminimized during operation of the apparatus of the present invention inthat the configuration of the cryogenic liquid nozzles form the sides ofan entrapment zone of droplets of cryogenic liquid into which the liquidproduct droplets are introduced. The entrapment zone is created byintroducing the cryogenic liquid into the housing in a downwardlydirected planar spray pattern, using at least three cryogenic liquidnozzles. The liquid product droplets are contained within the entrapmentzone, and directed in a downward direction such that the liquid productdroplets do not impinge upon or break through the walls of theentrapment zone.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a perspective view, partially cut away, of an upper portion ofan apparatus constructed in accordance with a preferred embodiment ofthis invention for producing frozen particles of a liquid;

FIG. 2 is a partially schematic representation of an apparatus forproducing frozen particles using the apparatus of FIG. 1;

FIG. 3 is a cross section of FIG. 1 taken along the section lines 3--3depicting the upper portion of the apparatus for producing frozenparticles;

FIGS. 4a, 4b, and 4c are partially schematic representations of variousviews of a cryogenic liquid nozzle;

FIG. 5 is another alternative embodiment of the invention depicting anUltrasonic nozzle which can be used as a liquid product nozzle in theapparatus of FIG. 1;

FIG. 6 is a bottom view of the cryogenic liquid nozzles showing the flatplanar spray pattern entrapment zone of this invention; and

FIG. 7 is a schematic representation of the cryogenic liquid nozzle andproduct liquid nozzle spray patterns used in this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION

There may be seen in FIGS. 1 and 2, preferred embodiment of an apparatusconstructed in accordance with this invention for producing frozenparticles of a liquid product, the apparatus comprises a generallycylindrical housing 6, a liquid product nozzle, such as an atomizationnozzle 8, for introducing droplets of the liquid to be frozen, and aplurality of cryogenic liquid nozzles 12 for introducing a cryogenicliquid into the housing 6 in a manner that directs the cryogenic liquidin a downward direction to form an entrapment zone or curtain cryogenicliquid droplets 36, whereby the cryogenic liquid freezes the liquidproduct droplets 63 to produce frozen particles.

FIGS. 1 and 3 depict an atomization nozzle 8 suitable for use as theliquid product nozzle of the present invention. Such atomization nozzles8 are known in the art and are available commercially. The atomizationnozzle 8 is directed downwardly into the housing 6 so as to dischargeliquid product in a vertically downward direction and designed so that agas from a source not shown, but depicted by arrow 20, such as nitrogenor air, is forced under pressure through an annular channel 60, whichsurrounds a centrally located tube 62, through which the liquid product,shown by arrow 18, is discharged under pressure, such that the gas 20breaks up the discharging liquid product into the liquid productdroplets 63. Nitrogen is the preferred inert gas for use in theatomization nozzle 8. The end of the atomization nozzle 8 through whichthe liquid product is discharged is tapered inwardly providing a thinannulus through which the gas escapes at high velocity disposed aboutthe liquid product. Such atomization nozzles 8 are generally useful forproducing droplets in the size range of about 8 to about 4300 μm.

Several examples of known types of droplet forming nozzles oratomization nozzles can be used as the liquid product nozzle, includingultrasonic nozzles, hydraulic nozzles, and pneumatic nozzles. An exampleof a suitable ultrasonic nozzle for use in the present invention is theModel 8700 nozzle available from Sono-Tek Corp., Poughkeepsie, N.Y. andan example of a suitable hydraulic nozzle is Model SU-5 available fromSpraying Systems Co. (Wheaton, Ill.). Pneumatic nozzles are preferred.

Referring to FIGS. 1 and 2, the preferred housing 6 has an upper plate30 and a vertically disposed cylinder 28 having a diameter larger thanthe diameter of the upper plate 30, the upper plate 30 and cylinder 28connected by a truncated conical section 26 having an upper conicaldiameter the same as the diameter of the upper plate 30 and a lowerconical diameter the same as the lower diameter of the cylinder 28.

The diameter of the upper plate 30 should be sufficiently large to allowfor the presence of a plurality of cryogenic liquid nozzles 12, theliquid product nozzle, such as an atomization nozzle 8, and a baffle 16(if used) between the plurality of cryogenic liquid nozzles 12 and theliquid product nozzle.

The truncated conical section 26 should preferably be designed so thatit is of sufficient length to allow for the formation of the entrapmentzone of cryogenic liquid droplets.

The lower diameter of the cylinder 28 is defined by the lower conicaldiameter of the truncated conical section 26 and the cylinder should beof sufficient length to enclose and direct the frozen product particlesand any gaseous cryogen which may have evaporated from the cryogenicliquid into the lower end of the cylinder 28 of the housing 6.

Preferably the apparatus is provided with a collection means 34 for thefrozen particles. Any collection means 34 large enough to capture andhold the frozen particles produced in the housing 6 can be usedincluding relatively simple devices such as trays which are manuallyoperated as well as fully automated conveyor type systems fortransporting the frozen particles away from the apparatus of theinvention. The collection means 34 should be kept at a temperature belowthe freezing point of the frozen particles to insure that the frozenparticles do not melt; such a temperature can be maintained, preferably,using the cooling effect resulting from the evaporation of cryogenicliquid to gaseous cryogen.

Preferably, a baffle 16 is included between the cryogenic liquid nozzles12 and the atomization nozzle 8. Such a baffle 16 serves to shield theliquid product nozzle 8 from the cryogenic liquid introduced into thehousing 6 by the cryogenic liquid nozzles 12. The baffle 16 can besupported by any means sufficient to hold it between the atomization 8and the cryogenic liquid nozzles 12 and should extend between theatomization nozzle 8 and the liquid cryogen nozzles 12 to a depthsufficient to shield the product nozzle 8.

By "liquid product" is meant any liquid which is to be frozen intouniform spherical particles. The preferred liquid products are aqueoussolutions useful as diagnostic reagents or pharmaceutical reagents,including but not limited to solutions containing dissolved proteinssuch as enzymes, antibodies, antigens, vitamins, and hormones, solutionsof other biological materials such as nucleic acids, antibiotics, andvarious drugs. Examples of such diagnostic reagents include aqueoussolutions of biologically active substances such as nicotinamide adeninedinucleotide (NAD), which can be used in toxicology testing foranalytically determining lactic acid and ethyl alcohol, and NAD-reduceddisodium salt trihydrate (NADH), which can be used in analyticallydetermining alpha-hydroxybutyrate dehydrogenase, the amount of which inturn can be related to the amount of the isoenzymes LD1 and LD2 oflactate dehydrogenase (LDH). These biologically active substances arepreferably combined with an excipient such as mannitol or trehalose, anda lubricant such as carbowax. Other examples of suitable liquid productsfor use as diagnostic reagents and which can be frozen in accordancewith the present invention include solutions of indicator compounds suchas the organic dye dichloroindophenol, useful in determiningpseudocholinesterase (PCHE), and liquid suspensions such as for example,a slurry of creatine kinase MB (CKMB) monoclonal antibody-coatedchromium dioxide particles suspended in water, which can be used to testfor the MB isoenzymes of creatine kinase.

One of the important aspects of the present invention is that frozenparticles produced using the apparatus and process of this inventionretain the biological and chemical characteristics and properties of theliquid product from which they are produced. For example, liquidproducts containing dissolved proteins such as enzymes can be frozenusing the present invention to produce particles which retain theenzymatic activity of the aqueous liquid product.

By "liquid product nozzle" is meant any nozzle which can be used tointroduce droplets of liquid product to be frozen into the apparatus ofthe present invention. The droplets of liquid product can vary dependingon the desired frozen particle size. The liquid product nozzle thereforeshould be chosen so as to provide for droplets of a size sufficient toprovide for the desired frozen particles. The preferred particle sizerange for the frozen particles produced using the apparatus of thepresent invention is about 75 to 600 μm.

Several types of cryogenic liquid nozzles are suitable for use with thepresent invention. A cryogenic liquid nozzle which produces a planarsuch as a flat or curved spray pattern is preferred. An example of asuitable nozzle for use as a cryogenic liquid nozzle is Model #HVV 9510,available from Spraying Systems, Inc.

One of the important features of the present invention is that thecryogenic liquid nozzles 12 are positioned so that they dischargecryogenic liquid to produce a spray pattern which forms an entrapmentzone or a curtain around the liquid product droplets 63. The entrapmentzone is defined by an interior region formed by a substantiallycontinuous, annular downwardly directed circumferential wall ofcryogenic liquid. At least 3 liquid nozzles are required to form theentrapment zone. The entrapment zone formed by 3 cryogenic liquidnozzles is in the shape of a triangle.

Preferably 4 to 6 cryogenic liquid nozzles are used, each nozzleproducing a downwardly directed planar spray pattern. The entrapmentzone produced using such a preferred spray pattern with four suchnozzles is in the shape of a box such as a square or rectangle. FIG. 6depicts such a configuration.

By forming an entrapment zone around the liquid product droplets 63 theapparatus of the present invention provides for a continuous high yieldmanufacturing process which can produce frozen particles of a uniformspherical size and homogeneous chemical composition which do not impingeagainst or adhere to the sides of the apparatus housing 6. The cryogenicliquid nozzle 12 has a slit shaped orifice which (FIG. 4a), is availablewith different slit sizes to discharge the cryogenic liquid as a flat orplanar spray pattern with the planar portion defining various sprayangles.

FIG. 7 illustrates the entrapment zone 97 around the spray solution 63formed by the cryogenic liquid spray 36.

By "housing" is meant a chamber in which the liquid product droplets arefrozen. The housing 6 confines any gaseous cryogen which may haveevaporated from the cryogenic liquid. The housing 6 should be vented toallow for the escape of any evaporated gaseous cryogen. A suitablehousing 6 for use with the present invention can have a variety ofshapes including rectangular, square, and cylindrical shapes. An upperportion of the housing 6 is preferably used for the introduction of thecryogenic liquid and the liquid product droplet 63, and for theformation of the frozen particles. A lower portion of the housing 6 ispreferably used for collecting the frozen particles and venting anygaseous cryogen which may have evaporated from the cryogenic liquid. Thehousing 6 need not be insulated; an uninsulated housing 6 is preferred.

The preferred configuration of the cryogenic liquid nozzles 12 is aplurality of four nozzles positioned in a ring manifold 10. Thepreferred cryogenic liquid nozzles 12 produce a downwardly directed flator planar spray pattern. The cryogenic liquid is introduced into thering manifold 10, shown by 22, through a tube 14. The use of a pluralityof nozzles screwed into a manifold for the cryogenic liquid nozzles 12provides for the introduction of flat streams of cryogenic liquid shownby 36 into the housing 6. These flat sprays of cryogenic liquid shouldbe sufficient to produce the maximum amount of frozen particles for agiven amount of liquid product and should provide for a uniformenvironment of liquid cryogenic droplets and gaseous cryogen within thehousing 6 of the apparatus in order to be useful for the continuousmanufacturing of frozen particles. The cryogenic liquid spray dropletsshould be sufficiently small so that cryogenic liquid does not run outthe bottom of the housing 6. The formation of an entrapment zoneproduced by a plurality of cryogenic liquid flat spray, is important forproviding for the optimal heat transfer between the cryogenic liquid andthe liquid product droplets 63.

Preferably, the temperature at the lower cylinder 28 outlet ismaintained at about -100° to about -180° C. This temperature range canbe obtained typically by choice of cryogenic liquid, adjustment ofcryogenic liquid flow rate, and the sizes and quantity of nozzles. Ifthis temperature range cannot be achieved, the liquid product flow ratescan be adjusted so that the liquid product flow rate into the apparatuscan be lowered.

Initially the liquid product flow rate can be adjusted to about 300milliliters per minute (ml/min) and the atomization gas pressure can beadjusted to about 11 pounds per square inch (psi). By adjusting liquidproduct flow rate and atomization pressure a desired particle size canbe achieved. For example, an undesirably small particle size can becorrected by lowering the atomization pressure. Other variables whichcan be optimized to achieve a desired particle size include cryogenicliquid flow rate, choice of type of liquid product and cryogenic liquidnozzles 12, and choice of housing 6.

FIG. 5 depicts a ultrasonic nozzle assembly 64 suitable for use as theliquid product nozzle of the present invention. Such ultrasonic nozzleassemblies 64 are known in the art and are commercially available.Ultrasonic nozzles produce droplets of the liquid product byelectrically generated vibrations of the nozzle and are typically usefulfor producing droplets in the size range of about 10 to about 100 μm.The ultrasonic nozzle assembly 64 has a nozzle 42 with an axiallyprojecting threaded boss 43 and a hollow core 49. A pair ofpiezoelectric crystals 38, each having an associated electrode 39,40 arereceived on the boss 43. The crystals 38 are held in place by threadingthe back piece 44 onto the boss 43. The nozzle 42, crystals 38,electrodes 39,40 and back piece assembly 44 is elastically supported inthe threadably engaging front housing 45 and rear housing 46 by thefront and rear O-rings, 47 and 48 respectively. An ultra high frequencyelectrical signal is supplied to the piezoelectric crystal discs 38,39through the connector 37 to the input electrode 39, with a ground beingprovided through electrode 40 to the ground lug 41. The electricalsignal causes the crystals 38,39 to expand and contract at theelectrical excitation frequency, thus calling the nozzle 42 and backpiece 44 to vibrate by virtue of their elastic mounting on O-rings 47and 48. Liquid product that is introduced into the hollow core 49absorbs some of the vibrational energy. The vibrational energy sets upwave motions in the liquid whose peaks become unstable and break awayfrom the liquid mass beneath, causing a fine mist at the tip of thenozzle 50.

By "cryogenic liquid" is meant any liquid capable of freezing the liquidspray of reagent into particles under atmospheric conditions. Variouscryogenic liquids which can be used in the present invention includenitrogen, carbon dioxide, argon and various fluorocarbons. Preferablythe cryogenic liquid used is nitrogen.

By "cryogenic liquid nozzle" is meant any nozzle for introducing acryogenic liquid into the apparatus of the present invention.

EXAMPLE 1 Construction of an Apparatus for Producing Frozen Particles ofa Liquid Product

An example of an apparatus designed and built for producing frozenparticles of a liquid product in accordance with the present inventionconsisted of two circular sections and a top plate 30 of FIG. 1 weldedto each other.

The lower section was a 14 inch (") diameter, 48" high open-endedvertically disposed lower cylinder FIG. 2 (28). The truncated conicalmiddle section, FIG. 2 (26), had a lower diameter of 14", an upperdiameter of 91/2", and a height of 10". The top plate, FIG. 1 (30),consisted of an 91/2" diameter disk with a 3" diameter hole in itscenter and a 1.5" baffle, FIGS. 1 and 3 (16), extending downward.

All of the sections including the top plate 30 of the vessel wereconstructed using 0.125" thick, 304 grade stainless steel and the entirevessel was mounted thru the top of a product collection box (5).

The cryogenic liquid nozzles, FIG. 1, (12), were nozzles screwed into ahollow ring manifold, FIG. 1 (10), with an inside diameter of 31/2", anoutside diameter of 6", and a 1" by 3/4" rectangular shaped crosssection using 0.187" thick 304 stainless steel. The cryogenic liquidnozzles (12) were 4 Spray Systems Model 9510, equally spaced around thebottom of a ring manifold, FIG. 1 (10). The nozzles 12 were disposedabout the atomization nozzle 8.

The cryogenic liquid was introduced into the housing 6 of the apparatusby forcing it through the hollow interior of the ring manifold 10 andout of the nozzles FIG. 1 (36).

Three equally spaced 1 inch long, 3/4 inch diameter vertical posts, FIG.1 (32), were attached to the top of the ring manifold 10 to support itwithin the housing 6. The bottom portion of the posts were welded to thering. The top portion of the posts were drilled and tapped for1/4-20×3/4 inch screws which provided a means for centrally mounting thering manifold 10 to the top plate 30 of the vessel. A conventional bulkhead fitting (not shown) was also attached to the top of the ringmanifold 10 which allowed a one-half inch diameter cryogenic liquid feedline, FIGS. 1 and 3 (14), to be attached to and supply cryogenic liquid,FIGS. 1, 2, and 3 (22), to the hollow interior of the ring manifold 10.

The liquid product nozzle was a pneumatic atomization nozzle FIG. 1 (8),with a 1.1 millimeter (mm) opening, (Model SU-5, Spraying Systems,Inc.). The atomization nozzle 8 was centrally mounted using a cross-barin the central hole of the upper plate 30 such that the tip of theatomization nozzle 8 was even with the tips of the cryogenic liquidnozzles (12). A peristaltic pump (pump model no. 7520-00, Master FlexCo.) was used to feed the liquid product to the atomization nozzle 8.

EXAMPLE 2 Producing Frozen Particles of A CKMB Diagnostic Reagent Usefulin a Creatine Kinase-MB (CKMB) Immunoassay

Into a 10 liter (L) stainless steel pot was added 1703 milliliters (ml)of deionized water. The following components were added, mixed, andallowed to dissolve in order: 360 trehalose (Sigma AF, St. Louis, Mo.),95 polyethylene glycol (PEG 8000) (Sigma AF, St. Louis, Mo.), 215 bovinealbumin (Miles Inc., Kankakee, Ill.), 252 sodium chloride (VWRScientific, Bridgeport, N.J.), 1.8 g magnesium chloride (VWR Scientific,Bridgeport, N.J.), 254 disodium PIPES buffer, (Research Organics, Inc.,Cleveland, Ohio), 39 PIPES (Research Organics Inc., Cleveland, Ohio),125 ml of a CKMB conjugate solution consisting of 1:1 ratio of F(ab')2anti-CKMB antibody fragments and β-galactosidase (prepared as describedbelow), and 1.1 g mouse IgG antibody (Scantibodies Laboratory, Santtee,Calif.) to eliminate non specific binding.

The cell lines producing the monoclonal antibodies employed wereobtained using the procedure described in U.S. Pat. No. 4,912,033 and inVaidya et al., Clin. Chem. 32(4): 657-663 (1986), the disclosures ofwhich are hereby incorporated by reference.

The anti-CKMB monoclonal antibodies so obtained were purified andisolated using affinity chromatography on Protein A Sepharose (PharmaciaFine Chemicals, Uppsala, Sweden). Protein A is a polypeptide (MW 42,000)isolated from Staphylococcus aureus which binds immunoglobin withoutinteracting with the antigen binding site.

While the above described method is preferred, monoclonal antibodies canbe purified using any number of standard techniques such as ammoniumsulfate precipitation dialysis, affinity chromatography, ion exchangechromatography etc. These and other methods for the isolation andpurification of monoclonal antibodies are described in general byGoding, Monoclonal Antibodies: Principles and Practice, Academic press,London and New York, 1983 and in U.S. Pat. No. 4,533,496 the disclosuresof which are hereby incorporated by reference.

The anti-CKMB monoclonal antibody used to produce the immunoreactivefragment described below was obtained as described above. The clonenumber was 2580 CC 4.2, and the monoclonal antibody was an IgG2bsubclass.

The purified anti-CKMB monoclonal antibody was dialyzed overnight at 4degrees C. (°C.) against an acetate buffer containing 100 millimolar(mM) sodium acetate and 150 mM sodium chloride, pH. 3.5. The dialyzedantibody solution was diluted to a concentration of 5 milligrams permilliliter (mg/ml) using the acetate buffer. The antibody solution wasplaced in a water bath at 37° C. for about 5 to 10 minutes.

A 10 mg/ml solution of pepsin (Sigma Chemical Co., St. Louis, Mo.) wasprepared in the acetate buffer. The amount of pepsin required to give aweight ratio of antibody to pepsin of 50:1 was determined and thedetermined amount of pepsin solution was added to the antibody solutionas the antibody solution was stirred. The mixture was incubated forabout 10-15 minutes. The reaction was then stopped by slowly adding 3.5molar (M) Tris base drop wise until the pH of the solution was in therange of 7.0 to 8.0. The resulting F(ab')2 preparation was then passedthrough 15-20 ml of Sepharose having Protein A bound to it in a 2.2×25centimeter (cm) column at a flow rate of about 4-4.5 ml per hour. Theprotein peak was monitored by recording the absorbance of the fractionsat 280 nm. The protein peak was collected and concentrated to about 30mg/ml using an Amicon stirred cell fitted with a 62 mm PM 30 membranefilter (both purchased from the Amicon Corp.). The sterilized F(ab'2)concentrate was filtered and stored at -20° C.

The sterilized F(ab')2 concentrate was coupled to β-galactosidaseconjugate using the procedure substantially as described by Kitagawa etal., Enzyme labeling with N-hydroxysuccinimidyl ester of maleimide in"Enzyme Immunoassays, Ishikawa et al., Eds., pp 81-90 (1981), thedisclosure of which is hereby incorporated by reference. Anti-CKMBmonoclonal antibody F(ab')2 fragment was dialyzed against an antibodydialysis buffer containing 20 mM phosphate buffer, 300 mM NaCl, pH 7.0).One mole of F(ab')2 was mixed with 30 moles of N-succinimidyl, 4(N-maleimido methyl) cyclohexane-1-carboxylate (SMCC) and incubated atroom temperature for 35 min. with constant stirring. The mixture wasloaded On a Sephadex G-25 column (2.2×13 cm) equipped with the UVdetector (absorbance 280 nm). The activated F(ab')2 fragment was elutedusing the antibody dialysis buffer. The protein peak was collected, itsvolume recorded and the protein concentration estimated. One mole of E.coli β-galactosidase (Boehringer Mannhelm) equivalent to 1 mole of SMCCactivated F(ab') 2 was dissolved in the antibody dialysis buffers.Activated F(ab')2 was mixed with β-galactosidase and incubated for atleast 25 minutes at 25° C. with constant stirring. Synthesis of theconjugate was monitored using an HPLC (LKB) equipped with a 100 μl loopGF 450 analytical column. The reaction was quenched when the leadingpeak extended beyond the second peak on the chromatogram by adding 10 μlof 0.1M N-ethylmaleimide solution for every ml of conjugate reactionmixture. The mixture was concentrated to 4.0 ml using an Amicon stircell and YM 100 filter (both purchased from the Amicon Corp.). Conjugateconcentrate was filtered through 0.2μ syringe filter and purified usingLKB HPLC equipped with 1 ml loop GF 450 column, UV monitor, fractioncollector and chart recorder. Appropriate fractions were collected andpooled and absorbance was measured at a wavelength of 280 nm to estimatethe protein concentration. The resulting concentrate was filtered,sterilized and stored at 40° C. Conjugate concentrate was diluted asneeded in a β -galactosidase conjugate dilution buffer (33.5 g PIPES(piperazine-N, N'-bis (2-ethanesulfonic acid)) 0.2 g MgCl₂, 29.2 g NaCl,100 g bovine serum albumin, and 0.167 g mouse IgG per liter of deionizedwater, pH 7.0) for the CKMB assay.

The solution was adjusted to a final volume of 4.8 L with deionizedwater and filtered through a 5 μm filter (Gelman Sciences, AnnarborMich. (product No. T 505141).

Liquid nitrogen from a 60,000 pound (lb.) tank was fed through aone-half inch diameter flexible steel tube at flow rate of 3.2 kilogramsper minute (Kg/min) to the ring manifold having the cryogenic liquidnozzles (12). Liquid nitrogen was thereby forced out of the cryogenicliquid nozzles. A steady state temperature measured centrally at theextreme bottom of the housing 6 was in the range of -150 to -170 degreesC. (°C.). An 18 inch square 2.0 inch deep collection tray FIG (2) 34 waspre-chilled to -35° C. and placed about 2" below the opening of thevessel.

Nitrogen gas at a pressure of 10 pounds per square inch (psi), FIGS. 1,2, and 3 (20), and at a flow rate of 300 milliliters per minute(ml/min), was fed to the atomization nozzle.

Frozen particles were collected in the collection tray 34 and manuallydistributed in the tray 34 to form a uniformly thick layer of frozenliquid product particles about 5/8" deep. The liquid product andnitrogen gas flows to the atomization nozzle were stopped and theproduction run of frozen particles was completed.

The average particle size was determined to be 402 microns by filteringthe particles through a series of known size sieves, with the sizedistribution shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Size (microns) % of total particles                                           ______________________________________                                        >590           16.4                                                           505-590        19.6                                                           335-504        25.2                                                           215-334        10.8                                                           165-215        7.6                                                            110-164        14.0                                                            75-109        6.4                                                            ______________________________________                                    

β-galactosidase activity of the above liquid product was measured bothbefore and after spray freezing using the aca® discrete clinicalanalyzer (E. I. dupont de Nemours and Company, Wilmington, Del. 19898).The pre spray freezing sample was obtained by diluting 1 mL of theliquid product with 20 mL of water. A post freezing sample was obtainedby dissolving 1 gm of the frozen liquid product in 4 mL of water anddiluting this solution with 80 mL of water. The prepared samples wereplaced in standard aca sample cups. The sample cups along with 3 acaMCKMB packs (standard aca® discrete clinical analyzer packs availablefrom E. I. Du Pont de Nemours and Company, Wilmington, Del.) were loadedon the aca. The aca dispensed 100 μl of sample, 2 mL of phosphatebuffer, pH 7.8, and 2.9 mL of water into each MCKMB pack. Breaker mixer1, a component of the aca® discrete clinical analyzer which breakstablet reagents in the pack and facilitates the mixing of the reagents,was utilized to dissolve and mix the pack reagents with the sample.Bound enzyme reacted with chlorophenol red galactoside (CPRG) containedin the pack reagents at 37° C. to form chlorophenol red (CPR). After 4.2minutes the absorbance of the contents of the pack was measured at 577and 600 nm wavelengths. 577 was the primary wavelength at which the CPRhas maximum absorbance, and 600 nm was the blanking wavelength. The 600nm reading was subtracted from the 577 reading to eliminate interferencedue to suspended particles and the resulting average absorbance numberwas multiplied by 20 due to the 1 to 20 dilution of the sample. Theresults, indicated minimal loss of enzymatic activity due to sprayfreezing. Thus, the usefulness of the present invention in producingfrozen particles of diagnostic reagents which retain their biologicalactivity upon freezing has been demonstrated.

What is claimed is:
 1. An apparatus for utilizing a cryogenic liquid forproducing frozen particles of a liquid product which comprises:(a) ahousing; (b) means positioned in the housing including at least threecryogenic liquid nozzles for producing a substantially continuous,annular downwardly directed circumferential wall of cryogenic liquiddefining an interior entrapment zone; (c) a liquid product nozzlepositioned in the housing for introducing droplets of the liquid productinto the entrapment zone whereby the cryogenic liquid freezes the liquidproduct droplets to product frozen particles; and (d) a baffle locatedbetween the liquid product nozzle and the plurality of cryogenic liquidnozzles to shield the liquid nozzle and to prevent freezing of theliquid product in the liquid nozzle.
 2. The apparatus of claim 1 whereinthe liquid product nozzle is an atomization nozzle.
 3. The apparatus ofclaim 1 wherein the liquid product nozzle is an ultrasonic nozzle. 4.The apparatus of claim 1 wherein the cryogenic liquid nozzles arepositioned in a ring manifold and produce a planar spray pattern.
 5. Theapparatus of claim 1 wherein the housing comprises an upper plate havingan upper diameter, and a vertically disposed cylinder having a diameterlarger than the upper diameter, the upper plate and the cylinderconnected by a truncated conical section.
 6. The apparatus of claim 1wherein the apparatus has a collection means below the liquid productnozzle for the frozen particles.
 7. The apparatus of claim 1 wherein theentrapment zone is box-shaped.
 8. A process for utilizing a cryogenicliquid for producing frozen particles of a liquid product in a housingwhich comprises the steps of:(a) introducing the cryogenic liquid intothe housing in an annular, downward direction creating an substantiallycontinuous downwardly directed circumferential wall of cryogenic liquid,defining an interior entrapment zone; and (b) introducing droplets ofthe liquid product into the entrapment zone, whereby the cryogenicliquid freezes the liquid product droplets to produce frozen particles.9. The process of claim 8 wherein the liquid product is an aqueoussolution.
 10. The process of claim 9 wherein the aqueous solutioncontains proteins.
 11. The process of claim 8 wherein the liquid productparticles have a diameter of about 75 to 590 microns.
 12. The process ofclaim 8 wherein the liquid product particles are spherical.
 13. Theprocess of claim 8 wherein the cryogenic liquid is applied by disposingthe cryogenic liquid circumferentially about the liquid productdroplets.